Behavioral imaging

The NL-2p1 microscope incorporates two imaging systems: a wide-field 'tail' camera for large-scale observation and a tissue camera for high-resolution specimen imaging through the objective. Achieving optimal contrast requires precise illumination control. Diffuse, wide-angle illumination in environments with reflective surfaces, such as the objective barrel and petri dish, can introduce glare and overexpose non-target areas. To address this, we developed a fiber optic light delivery system. This system, utilizing optimized LED optics and custom 3D-printed components, provides a cost-effective, high-quality illumination solution.

Empirical observations indicate that optimal behavioral camera illumination, particularly for tail movement observations, is achieved by directing light from above and laterally onto the fish. Conversely, tissue camera imaging benefits from illumination directed from below. Simultaneous illumination from both optical pathways, using the same wavelength, significantly degrades image quality in each channel due to non-selective illumination. To mitigate this interference, we employ distinct wavelengths for each imaging pathway. Specifically, since there is already an established system of filters in the red light arm of the PMT system, leading to the tissue camera, especially the 801 nm SP dichroic positioned in the scanning path of the microscope (see PMT detection system), we chose the 780 nm center-wavelength LED for the tissue camera illumination. For the wide-angle imaging we opted for the 815 nm center-wavelength LED. Their spectra are shown in the figure below.

The image below illustrates the components of this illumination setup (excluding the LED light sources located outside of the microscope enclosure). The '815 nm' label indicates the optical fiber delivering light from an 815 nm center-wavelength LED, used for the wide-field camera. The '780 nm' label identifies the fiber supplying illumination from a 780 nm center-wavelength LED, specifically for the tissue camera.

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For detailed information on individual components, see